1. Field of the Invention
This invention relates to an apparatus and a method for measuring binding between two molecular components resembling biologically active molecules or fragments thereof, such as and without limitation, binding between a specific protein and a specific nucleotide or G-protein. In one embodiment, the affinity of said binding is modulated by the binding of one molecular component to a third molecule. It is the sense of the present invention that said third molecule does not compete with binding between the first two components, but rather said third molecule induces a change in one of the first two components which results in a change in affinity between said molecular components. In another embodiment, measurement of binding between a protein component and its intended DNA response element is in itself of value. The apparatus and method of the present invention are of a type known by those skilled in the art as evanescent sensor fluorometry, and represents an improvement upon the apparatus and method described in U.S. patent application Ser. No. 08/616,576 entitled Surface Treatment and Light Injection Method and Apparatus which is assigned to the assignee of the present invention. As such, the apparatus described in the present invention can be equally well applied to immunoassay, which was the application toward which the apparatus of the previous patent application was directed.
The method of the present invention has particular relevance to study of the effect of certain test compounds, such as and without limitation, hormone mimics, on biological signal transduction which is mediated by binding of biological receptors and/or regulatory molecules to subsequent molecules such as and without limitation DNA molecules, involved in the transduction mechanism. The word "receptor" is defined for purpose of this invention according to the definition appearing in Illustrated Dictionary of Immunology, edited by Julius M. Cruse and Robert E. Lewis and published by CRC Press, Boca Raton, 1995, p.258, ISBN 0-8493-4557-X: "A molecular configuration on a cell or macromolecule that combines with molecules that are complementary to it." The term "regulatory molecule" is defined "a molecule which, upon binding to a specific complementary molecule, initiates a sequence of events resulting in regulation of a biological process."
In a first embodiment, the apparatus and method utilizing the principles of the invention are adapted for use as a screening tool for recognizing the presence of estrogen mimics in a sample. In a second embodiment the apparatus and method utilizing the principles of the invention are adapted for measuring estrogen receptor content in a tissue biopsy sample and evaluating in vitro the probable response of cancer cells, of a type present in that tissue biopsy sample, to certain pharmacologic agents which act through receptor binding. In a third embodiment, the apparatus and method utilizing the principles of the invention are adapted for evaluating the competency of the p53 protein present in a tissue sample.
2. Background to the Invention
Many biological processes are regulated by the binding of regulatory molecules such as hormones, neurotransmitters or cytokines to specific biological receptor molecules. Upon binding to the regulatory molecule, the receptor activates the next step in a signal transduction mechanism by itself binding to another molecular component of the transduction mechanism such as a nuclear response element or G-protein. The affinity with which this second stage of receptor binding occurs, or in some cases, whether or not this second stage binding occurs at all, is affected by the binding of the regulatory molecule to the receptor. A review of such mechanisms can be found in an article entitled "Mechanisms of Signal Transduction: Sex Hormones, Their Receptors and Clinical Utility" by James L. Wittliff and Wolfgang Raffelsberger, which appeared in Journal of Clinical Ligand Assay, Volume 18, Number 4, Winter, 1995. This text is fully and completely incorporated herein by reference, word for word and paragraph for paragraph.
There are many benefits which derive from the study of both the binding of receptors to regulatory molecules and the second stage binding of the receptors to another component of the signal transduction mechanism. Such study can assist in the design of drugs which exert their biological effect through binding to biological receptors. It can also lead to recognition of compounds in the environment which have the capacity to disrupt important biological regulatory mechanisms by virtue of the ability of such molecules to bind to molecular receptors. It is believed that the presence of such molecules in the environment plays a role in the development of a variety of disease types including cancer, immune dysfunctions, and reproductive problems.
Current methods used for studying these binding phenomena are described in the previously cited review. Because the methods require physical separation of bound from unbound molecules, the methods are quite time consuming and do not have the capacity to provide real-time data while binding is occurring between a receptor and a regulatory molecule or between receptors and another component of the signal transduction mechanism. The reliance of current methods on radiolabeled ligands also limits the circumstances under which such measurements can be made. The apparatus and method of the present invention overcomes the limitations of the prior art by removing the need to separate bound from unbound molecules prior to performing a measurement, with the consequence that real time binding between components can be monitored and association and dissociation constants and equilibrium constants can be calculated far more quickly and easily.
The apparatus of the invention is a type of evanescent fiber optic sensor. Evanescent fiber optic sensors provide a method whereby a molecule bearing a fluorescent tag can be directly monitored as it binds to a binding partner attached to an optical fiber. Light traveling through an optical fiber at or near the critical angle is totally internally reflected so that it does not excite fluorescence in the surrounding solution. Total internal reflection does, however, produce an evanescent field which extends about 1000 angstroms from the surface of the fiber. This means that fluorescence of molecules binding to the surface of the fiber can be excited without exciting fluorescence of unbound molecules in the surrounding solution. Therefore measurement of binding can be made without the necessity for physical separation of bound from unbound molecules. Evanescent sensors based upon measurement at a certain time of fluorescent antigen bound to antibodies on the fiber have been used to perform immunoassays by calculating concentrations of antigen in a solution. These have been reported in literature and patents and are thoroughly described in the book Biosensors with Fiber Optics, Donald L. Wise and Lemuel B. Wingard, Jr. Editors; Humana Press, Clifton, N.J., 1991. This text is fully and completely incorporated herein by reference, word for word and paragraph for paragraph. The immunoassay-based evanescent sensors of the prior art do not utilize data collected continuously by the sensor over a time period to perform the assay. Rather a single point in time is defined for taking a single measurement from the sensor and a standard curve is prepared relating such single point measurements to concentration of antigen in the solution. The prior art is therefore directed toward assay of a specific compound in a sample rather than assessment of the kinetic and binding parameters describing the interaction between a component in the sample and a component attached to the sensor waveguide surface.
3. Background to the Evanescent Sensor Apparatus of the Invention
The essential feature of an evanescent biosensor, is confinement of the measurement area to the surface of the waveguide by taking advantage of the evanescent field associated with total internal reflection within the fiber. This was originally described in the context of immunoassay by Tomas Hirshfield in U.S. Pat. No. 4,447,546 entitled "Fluorescent immunoassay employing optical fiber in a capillary tube" which is herein incorporated by reference, line by line and word for word. The manner in which this functions is as follows.
Consider light incident at angle .theta. on the boundary between two optical media with indexes of refraction N and n (N&gt;n). When the light is incident on the boundary at angles greater than or equal to the critical angle, .theta..sub.crit where sin(.theta..sub.crit)=n/N, the light will be totally reflected from the surface. Although, light is not transmitted past the boundary and into the media with the lower index of refraction, electromagnetic theory shows that an evanescent electromagnetic field decays exponentially with perpendicular distance from the boundary. The characteristic l/e depth of this decay for light of wavelength .lambda. incident at angle .theta. is given by the equation: EQU (.lambda./4.pi.)(N.sup.2 sin.sup.2 .theta.-n.sup.2).sup.-1/2. Equation 1
This distance is large compared with the dimensions of proteins and biologically significant nucleotides. Thus, the light with wavelength .lambda..sub.1 will interact with fluorescent molecules, which are associated with any proteins or nucleotides that are attached near the probe's surface, to generate fluorescence at wavelength .lambda..sub.2. Because the waveguide is very large compared with the size of the proteins or nucleotides, a large fraction of the emitted fluorescence light at wavelength .lambda..sub.2 will intersect the fiber optic sensor, then be trapped inside due to total internal reflection, and finally be carried back to a solid state light detector in the control unit.
Prior designs of evanescent sensor instruments achieve delivery of excitation light to and collection of fluorescence from the sensor fiber by means of free space propagation from a focusing lens into the fiber sensing element without the use of an intermediate low loss beam shaping means (U.S. Pat. No. 4,608,344, Method for the Determination of Species in Solution with an Optical Wave-Guide, Carter, J. N., Dahne, C. and Place, J. F.), (U.S. Pat. No. 4,447,546, Fluorescent Immunoassay Employing Optical Fiber in Capillary Tube, Hirschefeld, T. E.), (Publication: Fluorometer and Tapered Fiber Optic Probes for Sensing in the Evanescent Wave, by Golden, J. P., Shriver-Lake, L. C., Anderson, G. P., Thompson, R. B., Ligler, F. S., in Optical Engineering, July, 1997, p. 1458-1462). Shaping of the entering excitation light into an annular beam is described in U.S. patent application Ser. No. 08/616,576 entitled Surface Treatment and Light Injection Method and Apparatus which is assigned to the assignee of the present invention describes injection of annularized light at or near the critical angle. All methods of the prior art require that each sensor cartridge be manually aligned with the light from the focusing lens by adjustment means such as and without limitation to x,y,z stages upon which the sensor cartridge is mounted or adjustment of the focusing lens. This requirement is not well adapted for use of the instrument by untrained personnel. Prior art also does not provide a means for preventing side bands from a laser source of excitation light, from entering the sensor. Prior art is plagued by the problem that light is lost from the fiber sensor at any point of contact which has a higher refractive index than that of the sample. Efforts to deal with this problem are described in several patents. U.S. Pat. No. 4,447,546, Fluorescent Immunoassay Employing Optical Fiber in Capillary Tube, Hirschefeld, T. E., 1984, holds the fiber in place using a supporting stopper out of siloxane and coating the ends of the fiber with a low refractive index silicone. This doesn't fully solve the problems because the refractive index of silicones and siloxanes is at best 1.367. For a fiber in an aqueous solution having refractive index of 1.33, this creates an NA, of about 30.1.degree.. Thus the light near the critical angle of 35.8.degree. will be lost in the siloxane.
A method for dealing with this difficulty of light loss due to improper matching of NA, is found in U.S. Pat. No. 5,061,857, Waveguide-binding sensor for use with assays, R. Thompson, and C. Villarruel, 1991. Here the sensor fiber is tapered so as to produce a transformation of the effective NA of the fiber. The teaching under that patent requires that the fiber be etched in hydrofluoric acid to achieve correct tapering, which creates problems with respect to manufacturability.
A third method for avoiding light loss where the fiber contacts a support is described in U.S. Pat. No. 4,671,938, Immunoassay Apparatus, T. A. Cook, 1987. In this teaching, the sensor fiber is held at its distil end, but not at its proximal end, thereby avoiding the issue of contact with the supporting structure. The direct injection of annularized light at or near the critical angle could not be accomplished under this prior teaching because the nature of the teaching precludes inserting the proximal end of the sensor fiber into the coupling capillary containing the annularizing fiber.
All reported prior art describing evanescent sensors involves chemical sensitization of optical waveguides having surfaces from which all materials other than the core material of the optical waveguide have first been completely and thoroughly removed, usually by treatment with strong acid. Methods employed in prior art to protect these sensors from the sensitivity degradation caused by the non-specific binding of biological proteins to sensor surfaces, requires exposing said sensitized sensor surfaces to a solution of non-interfering proteins so that the non-interfering proteins bind to said sensor surfaces to prevent the subsequent binding of the interfering proteins. Because this method is never completely effective and non-specific binding severely degrades the attainable performance of sensors described in prior art. However, prior art indicates that enhanced protection of surfaces from biological proteins is possible by completely covering surfaces with protective coatings. For example, methods have been employed to protect surfaces from nonspecific binding to materials used in implantable devices such as catheters or materials for prostheses. In that context, the amorphous copolymers of tetrafluoroethylene and bis-2,2-trifluoromethyl-4.5-difluoro-1,2-dioxole sold under the trademark TEFLON AF.RTM. has been dissolved in a solvent containing fluorinated alkanes such as FLUORINERT.RTM., and applied by deposition as a thin protective, totally enclosing layer to the surface of polymers in order to reduce thrombogenicity and complement activation. This is described in U.S. Pat. No. 5,356,668 by Duncan M. Paton, Timothy R. Ashton and Roshan Maini, 1994, entitled Fluorinating Polymer Surfaces.
U.S. patent application Ser. No. 08/616,576 entitled Surface Treatment and Light Injection Method and Apparatus which is assigned to the assignee of the present invention describes a method by which the nonspecific binding protection conferred by the copolymer of tetrafluoroethylene and bis-2,2-trifluoromethyl-4.5-difluoro-1,2-dioxole may be preserved, while still retaining a waveguide surface capable of chemical sensitization. A method is described therein showing a means to modify glass or silicon surface adhesion properties to substantially protect these surfaces from the non-specific binding of proteins by starting with a glass or silicon surface which has been coated with amorphous copolymers of tetrafluoroethylene and bis-2,2-trifluoromethyl-4.5-difluoro-1,2-dioxole, e.g. TEFLON AF.RTM. which have been baked onto the surface at temperatures near the copolymer's glass point so as to improve surface adhesion of the copolymers, and then using a solvent containing fluorinated alkanes such as FLUORINERT.RTM., to substantially remove all of said coating material from said surface, except for nearly undetectable trace amounts of surface contamination from constituents from said cladding which are visible using atomic force microscopy as an open network of elevated regions surrounding the underlying clean, bare, glass or silicon surface regions. That patent application does not describe means by which said treatment may be applied to fibers in a batch process so as to produce optical waveguides of uniform quality.
This same coating material of amorphous copolymers of tetrafluoroethylene and bis-2,2-trifluoromethyl-4.5-difluoro-1,2-dioxole, e.g. TEFLON AF.RTM., is applied to and baked on silica fibers for use as cladding on commercially available optical fibers which can be obtained from suppliers such as but not limited to Polymicro Technologies, Inc., 18019 N. 25th Ave., Phoenix, Ariz. 85023. However, in order for fiber having this cladding material to be used in an evanescentsensor, the said cladding must be removed from surface regions which will be chemically sensitized.